Azadirachtin Content Research Articles

Effect of azadirachtin applied systemically through roots of plants on the mortality, development and fecundity of the cabbage aphid (Brevicoryne brassicae)

The effect of low concentrations of azadirachtin applied systemically through root tissues of rape plants (Brassica napus subsp.napus) on the mortality, period of development, longevity and fecundity of the cabbage aphid (Brevicoryne brassicae L.) (Sternorrhyncha: Aphididae) was studied. The concentrations of azadirachtin A tested were: 0.25, 0.05, 0.005, 0.0005 and 0.00005 mg ml−1. The mortality of cabbage aphid nymphs, particularly during the period of ecdysis, increased significantly with increasing concentrations. Azadirachtin had no effect on the length of the development period of the respective immature stages of cabbage aphids. The average longevity of the cabbage aphids decreased with increasing azadirachtin concentration and time spent feeding. The fecundity of aphids decreased after the application of azadirachtin.

Effects of neem‐treated aphids as food/hosts on their predators and parasitoids

Abstract: Experiments were conducted in the laboratory and in greenhouses. Of three neem preparations sprayed upon eggs, only neem oil (NO) exerted a negative impact on the hatching rate of Coccinella septempunctata and Chrysoperla carnea. First instar larvae of Episyrphus balteatus proved to be highly susceptible, when feeding 24 h on aphids sprayed with neem kernel water extract (NKWE). First instar larvae of C. septempunctata showed a very high mortality when feeding on aphids sprayed with different neem preparations. Aphid feeding and live span was reduced. When NKWE had been applied to the soil, the mortality of larvae of E. balteatus and C. septempunctata were lower, when feeding on aphids. Second instar larvae of C. septempunctata were far less susceptible when feeding 48 h on neem‐sprayed aphids than first instars; the time of their development was prolonged, and aphid consumption reduced. Larvae of C. carnea proved to be less susceptible, when feeding on neem‐sprayed aphids, than E. balteatus and C. septempunctata. In C. carnea, however, significant influences were also observed in aphid consumption, time of development, mortality, longevity, and rate of deformity. NO, containing a very low concentration of azadirachtin A, had stronger negative effects than NeemAzal‐T/S®, in all observations. In the parasitoid Diaeretiella rapae, NKWE application to the soil induced negative reactions, when aphids on these plants were parasitized: low percentage parasitization, lowered mummy weight, low emergence rate of adults of F1 and even of F2. Foliar sprays of NKWE had less severe effects in this parasitoid species. The results are discussed with regard to their theoretical and practical significance.

Variability in Neem (Azadirachta indica) with respect to azadirachtin content.

There is a controversy over variations in azadirachtin content in neem (Azadirachta indica) seeds among various provenances and countries. Also, variations in azadirachtins are usually attributed to climatic conditions such as temperature and humidity. The present study was undertaken to evaluate qualitative and quantitative variability in azadirachtins A and B among various neem provenances or individual neem trees. Forty-three provenances of India were examined for intraprovenance variability in azadirachtin A and B content and oil percentage. Twenty-eight individual neem trees from five provenances of different agroclimatic regions were also examined for interprovenance variability. The azadirachtins were quantified using reversed phase analytical HPLC. There were wide variations in oil and azadirachtin contents among different provenances. Azadirachtin A ranged from 556.9 to 3030.8 mg kg(-)(1) of kernels, whereas azadirachtin B was in the range 43.1-590.6 mg kg(-)(1) of kernel among the provenances investigated. Analysis of variance among various neem provenances showed significant differences in oil content, azadirachtin A, total azadirachtin (A + B), and A:B ratio. There were individuals with high and low azadirachtins within a single provenance, and this trend was observed in all of the provenances selected from five agroclimatic regions of the country. Variations among individual trees of a particular provenance indicated that climatic factors such as rainfall, humidity, or temperature did not influence azadirachtin content in the neem trees. The present study shows that there are individual genetic differences among neem trees. A systematic study for tree improvement with a population of mother trees with desired traits should be undertaken by performing half-sib progeny trials and further selections by clonal propagations. The role of genetic makeup needs further research.

Determination of azadirachtin and fatty acid methyl esters of Azadirachta indica seeds by HPLC and GLC.

A simple and economical method has been developed to estimate the azadirachtin content and fatty acid composition of neem kernels. Neem kernels are crushed and soaked overnight in ethanol. The extract obtained is analysed by HPLC after filtering through a 0.22 micro m membrane. The peaks are separated using acetonitrile-water (40:60) 1 mL min(-1) as the mobile phase on an RP-18 column and monitored at 214 nm. For the determination of fatty acid composition, the fatty acids are directly transmethylated in the kernel powder by heating with methanol-acetyl chloride-benzene (20:1:4, v/v) for 1 h in a water bath. The fatty acid methyl esters (FAMEs) obtained are extracted in hexane and analysed using GLC. The separation of the FAMEs is achieved using an RH-Wax column using temperature programming, 170-200 degrees C at 2 degrees min(-1). The peaks are detected using an FID. Both the methods do not require any clean up or defatting of seeds. This results in faster, easier and more economical sample preparation.

Molecular analysis of micropropagated neem plants using aflp markers for ascertaining clonal fidelity

The importance of neem (Azadirachta indica A. Juss.) as a medicinal tree species has been acknowledged worldwide. Superior trees with desired traits such as high azadirachtin content have been identified and micropropagated. Somaclonal variants that may arise in vitro, however, pose limitations to large-scale micropropagation. It is, therefore, imperative to establish genetic uniformity of such plantlets by ensuring strict quality checks at various stages of in vitro culture. This is the first study that evaluates the applicability of amplified fragment length polymorphism (AFLP) markers in establishing clonal fidelity of tissue culture(TC)-raised neem plants. Seven AFLP primer combinations generated a total of 334 amplified fragments across the mother plant, TC progenies, and other neem accessions that were included as controls. Two hundred and thirty-nine amplified fragments were monomorphic across the mother tree and its TC progenies. No extra band was detected in the TC plantlets that was absent in the mother tree, indicating that the TC plantlets regenerated through nodal explants are indeed true-to-type. Ninety-five AFLP fragments were detected in the controls, which allowed their discrimination from the elite mother tree and its TC progenies. Similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother tree and its TC plantlets was ‘1’, indicating perfect similarity. Phenetic dendrogram based on UPGMA (unweighted pair group method of arithmetic averages) analysis further confirmed the true-to-type nature of TC progenies, since a tie was observed between the mother tree and its TC plantlets. On the contrary, the control neem accessions were distinct from the mother and its TC progenies. AFLP markers proved to be an ideal tool for routine analysis and certification of genetic fidelity of micropropagated plants prior to commercialization, especially in tree species because of their long generation time.

Community-level disruptions among zooplankton of pond mesocosms treated with a neem (azadirachtin) insecticide

A natural, plant-derived insecticide, neem, is being evaluated as an alternative insect pest control product for forestry in Canada. As part of the process to investigate the environmental safety of neem-based insecticides, a mesocosm experiment was conducted to assess the effects of neem on natural zooplankton communities. Replicate ( n=5), shallow (<1 m) forest pond enclosures were treated with Neemix® 4.5, at concentrations of 0.035 (the expected environmental concentration), 0.18, 0.70, and 1.75 mg/l active ingredient, azadirachtin. Zooplankton communities were quantitatively sampled over a 4-month experimental period in treated and control enclosures, and water samples were collected to track azadirachtin concentrations. Concentrations in water declined linearly with estimated DT 50 values of 25–29 days. Trends in abundance over time among populations of cladocerans, copepods, and rotifers were found to differ significantly among treatments. At the two highest test concentrations, adverse effects were obvious with significant reductions in several cladoceran species, and near elimination of the three major copepod species present. More subtle effects at the two lowest test concentrations were determined by comparing the community structure of enclosures across treatment levels and over time through an analytical process based on the multivariate statistical software, PRIMER. Significant effects on community structure were detected at both of these lower concentrations, including the expected environmental concentration of 0.035 mg/l azadirachtin. Differential responses among species (some increases, some decreases) caused detectable disruptions in community structure among zooplankton of treated enclosures. Perturbations to zooplankton communities were sufficient to cause measurable differences in system-level metabolism (midday dissolved oxygen concentrations) at all but the lowest test concentration.

Comparative toxicity of two azadirachtin‐based neem pesticides toDaphnia pulex

Azadirachtin (AZA)-based pesticides (Neemix and Bioneem) demonstrated toxicity in 48-h nonrenewal toxicity assays using Daphnia pulex at levels that were comparable with several organophosphate pesticides. The median lethal concentration (LC50) values for the two neem pesticides were found to be 0.028 and 0.033 microl/ml, respectively. The LC50 value for nonformulated (95% pure) AZA was determined to be 0.382 microg AZA/ml. Neemix and Bioneem were exposed to air and northern sky daylight in a light box at 24 and 37 degrees C for 1, 3, 6, and 9 d. Standard 48-h acute toxicity tests were used to determine the effect of aging in these dry environmental conditions. Neemix and Bioneem were also fractionated into volatile and nonvolatile fractions, and the toxicity of each was tested. Compared with Neemix, Bioneem remained toxic longer when exposed to light and air at 37 degrees C, indicating that this pesticide may be less prone to environmental degradation. When fractionated, the nonvolatile fractions for both pesticides exhibited significantly lower LC50 values than the full formulations. These results suggest that, depending on the application rate and environmental fate, AZA-based pesticides may have direct adverse effects on aquatic organisms and that the toxicity and stability of formulated pesticides depend on factors other than only the AZA concentration.

Neem the source of versatile chemicals, Azadirachtin in modern pest management: a review

Neem is known for its therapeutic and pesticidal applications. Azadirachtin is the chemical constituent in neem seed kernel which is active against wide spectrum of insect pests. Beside azadirachtin several other chemical constituents such as salannin, meliantriol, etc., are also known to possess biocidal activity. The azadirachtin content in seed kernel generally varies between 0.1% to 0.7% and is determined using reverse phase high-pressure liquid chromatography (HPLC) system. For commercial application azadirachtin content 1500 ppm in extract-based and 300 ppm in oil-based products is an important requirement. Almost all parts of the neem tree are known for its medicinal value. Several neem compounds such as nimbin, nimbidin, nimbinin, etc., are found active for various medicinal applications.

Multivariate Calibration for the Determination of Total Azadirachtin-Related Limonoids and Simple Terpenoids in Neem Extracts Using Vanillin Assay

Two-component and multivariate calibration techniques were developed for the simultaneous quantification of total azadirachtin-related limonoids (AZRL) and simple terpenoids (ST) in neem extracts using vanillin assay. A mathematical modeling method was also developed to aid in the analysis of the spectra and to simplify the calculations. The mathematical models were used in a two-component calibration (using azadirachtin and limonene as standards) for samples containing mainly limonoids and terpenoids (such as neem seed kernel extracts). However, for the extracts from other parts of neem, such as neem leaf, a multivariate calibration was necessary to eliminate the possible interference from phenolics and other components in order to obtain the accurate content of AZRL and ST. It was demonstrated that the accuracy of the vanillin assay in predicting the content of azadirachtin in a model mixture containing limonene (25% w/w) can be improved from 50% overestimation to 95% accuracy using the two-component calibration, while predicting the content of limonene with 98% accuracy. Both calibration techniques were applied to estimate the content of AZRL and ST in different parts of the neem plant. The results of this study indicated that the relative content of limonoids was much higher than that of the terpenoids in all parts of the neem plant studied.

Community‐level responses by stream insects to neem products containing azadirachtin

AbstractOutdoors sream channels weret teated with a commercial neem formulation, Neemixx® 4.5, and a neem extract (no formulation ingredients) to determine the effects on aquatic insect communities. An exposure period of 5 h was chosen to simulate the transient nature of pesticide residues in streams and rivers after aerial applications. Multivariate analytic procedures based on Bray‐Curtis similarity matrices were used to compare community structure among the replicate channels 9 d after treatment. No significant differences in community structure were found among controls and channels treated with Neemix at an azadirachtin concentration of 0.28 mg/L, but a multivariate measure of community stress indicated an increase in variability among treated channels. Significant differences in community structure were found among controls and channels treated with Neemix at 0.84 and 2.54 mg/L, and this resulted from reductions in several key taxa. During subsequent experiments with a neem powder extract, the formulation ingredients of Neemix were at least partially responsible for the significant effects on community structure at 0.84 mg/L azadirachtin. No significant differences were found among controls and channels treated with the extract at 0.9 mg/L, whereas the community structure of aquatic insects in channels treated at 3.0 mg/L differed significantly from controls. In a Canadian forest pest‐management context, the expected environmental concentration in water bodies of areas sprayed with azadirachtin at 50 g/ha is 0.035 mg/L.

Toxicity to the snailLimnaea acuminata of plant-derived molluscicides in combination with synergists

The effects of combinations of plant-derived molluscicides with piperonyl butoxide (PB) or ENT 8184 (MGK-264) were studied on different enzyme activities and biogenic amine levels in the nervous tissue of Limnaea acuminata. 24-h in vivo exposure of the nervous tissue of L acuminata to 40% and 80% of the 24-h LC50 of the plant-derived molluscicides Azadirachta indica oil, Allium sativum powder and oleoresin of Zingiber officinale rhizome and their active molluscicidal components azadirachtin, allicin and [6]-gingerol, alone or in combination (1 + 5) with PB or ENT 8184, caused a significant reduction in the activity of acetylcholinesterase, lactic dehydrogenase, acid and alkaline phosphatases and sodium-potassium ATPase. There was a significant increase in the activity of succinic dehydrogenase. In vivo exposure for 24 h to sub-lethal concentrations of azadirachtin, allicin and [6]-gingerol, singly and with PB or ENT 8184, significantly altered the dopamine and 5-hydroxytryptamine levels in the nervous tissue of L acuminata. It is concluded that these active moieties singly or with PB or ENT 8184 adversely affect all known neurotransmission mechanisms in the snail, either separately or through a complex interaction between the different neurotransmitters. © 2000 Society of Chemical Industry

Extraction and colorimetric determination of azadirachtin-related limonoids in neem seed kernel.

A colorimetric method was developed for the determination of total azadirachtin-related limonoids (AZRL) in neem seed kernel extracts. The method employed acidified vanillin solution in methanol for the colorization of the standard azadirachtin or neem seed kernel extracts in dichloromethane. Through the investigation of various factors influencing the sensitivity of detection, such as the concentration of vanillin, acid, and the time required for the formation of color, optimum conditions were selected to perform the assay. Under the optimum conditions, a good linearity was found between the absorbance at 577 nm and the concentration of standard azadirachtin solution in the range of 0.01-0.10 mg/mL. In addition, different extraction procedures were evaluated using the vanillin assay. The HPLC analysis of the extracts indicated that if the extractions were performed in methanol followed by partitioning in dichloromethane, approximately 50% of the value determined by the vanillin assay represents azadirachtin content.

Extracellular and intracellular actions of azadirachtin on the electrophysiological properties of cultured rat DRG neurones

The distinct electrophysiological actions of extracellular and intracellular azadirachtin (type A) were investigated using cultured dorsal root ganglion neurones from neonatal rats and the whole cell variant of the patch clamp technique. The cultured mammalian neurones were relatively insensitive to azadirachtin compared with some invertebrate preparations, such as insect chemosensory systems. Insect preparations have been found to respond to 1–100 nM azadirachtin. However, at concentrations of azadirachtin between 10 and 100 μM significant changes in the electrophysiological properties of cultured DRG neurones were seen. Extracellular application of azadirachtin prolonged the late repolarization phase of evoked action potentials and consistent with this produced modulation of voltage-activated K + currents. This modulation involved an initial transient increase in K + current followed by reversible inhibition when azadirachtin was applied at a concentration of 10 μM. Higher concentrations of azadirachtin (50 and 100 μM), had only inhibitory effects on voltage-activated K + currents. Azadirachtin produced a degree of voltage-dependent inhibition, with a greater level of K + current inhibition observed when neurones were held at −90 mV compared with −40 mV. Prolonged application of azadirachtin for 24 h reversibly reduced action potential amplitude. In contrast intracellular application of 100 μM azadirachtin via the patch pipette solution had no significant effects on action potentials but produced an increase in conductance. Intracellular azadirachtin activated several conductances including a TEA-sensitive K + current and an inward current that had an estimated reversal potential close to 0 mV. In conclusion, data showed that azadirachtin had different effects when it was applied inside and outside the neurones and that azadirachtin at high concentrations reversibly altered neuronal excitability mainly by modulating potassium conductances. It appears that rat cultured DRG neurones are less affected by azadirachtin than some insect sensory systems.

Oviposition bioassay responses of Culex tarsalis and Culex quinquefasciatus to neem products containing azadirachtin

AbstractNeem (Azadirachta indica A. Juss) products have been shown to exert pesticidal properties against a variety of insect species. In mosquito control programs, such products may have the potential to be used successfully as larvicides. In exploring other advantages of neem products, we studied the oviposition responses of Culex tarsalis Coquillett and Cx. quinquefasciatus Say to two experimental azadirachtin (AZ) formulations, wettable powder Azad™ WP10 (WP) and emulsifiable concentrate Azad™ EC4.5 (EC). Gravid Cx. tarsalis exhibited a distinct preference for the neem suspension of the WP, where significantly more egg rafts were collected from the treatment than from the control. The minimum effective AZ concentration for this activity was 0.5 ppm. The aged suspensions from 1–7 days at 0.5 and 1 ppm AZ were more active in eliciting oviposition responses in Cx. tarsalis than the fresh preparations. This activity of the aged suspensions lasted up to 14 and 21 days at 0.5 and 1 ppm AZ, respectively. Negative ovipositional responses were indicated in the tests of the EC vs. Cx. tarsalis, as well as both neem formulations vs. Cx. quinquefasciatus. In the tests of the EC formulation, significantly less gravid females were trapped by oviposition cups in the treatment than in the control, and in the tests of the WP significantly less egg rafts were collected from the treatment than from the control. The minimum effective concentrations for oviposition avoidance activity were 5 ppm AZ for Cx. tarsalis and 10 ppm AZ for Cx. quinquefasciatus, which lasted up to 1 and 4 days for these two species respectively. Neem products potentially used as mosquito larvicides may have many additional benefits in mosquito control programs, the oviposition modification noted in the current studies is one such example.

Insect growth regulating and antifeedant effects of neem extracts and azadirachtin on two aphid species of ornamental plants

Leaf disc choice test bioassay demonstrated that formulated neem seed extracts were highly deterrent and growth regulatory to rose aphid,Microsiphum rosae (L.) and Chrysanthemum aphid,Macrosiphoniella sanbornii (Gillete). Effective concentrations to produce 50% feeding deterrence was 0.80 and 0.84% respectively for 2nd instar nymphs irrespective of bioassay duration. The disruption of aphid feeding was related to the presence of azadirachtin concentration in the extract. The toxicity on contact from the leaf surface or via topical application due to azadirachtin was significantly different and topical treatment was at least 7 times more effective for both species. Thus growth regulatory effects of azadirachtin were influenced by the host plant and the stage of treatment. Field evaluation with formulated neem extracts revealed the effect to be more of growth regulatory nature thereby showing that azadirachtin is a physiological toxin for aphid species. Neem seed extracts reduced the population of aphid on respective host plants significantly, EC50 values being 0.88 and 0.96% forM. rosae andM. sanbornii respectively.

Effects of a Neem Compound on the Fecundity and Longevity of Ceratitis capitata (Diptera: Tephritidae)

A commercial formulation of neem was tested on adult Ceratitis capitata (Wiedemann) to determine its effects on fecundity and longevity. Four no-choice tests were carried out in the laboratory at constant conditions of temperature, humidity, and photoperiod. Different concentrations were tested mixing neem solutions with the adult’s rearing diet. The laboratory tests showed that neem significantly reduced fecundity by interfering with oogenesis, which resulted in a complete and irreversible sterility of females. A slight reduction of longevity was also observed. The number of eggs laid decreased with increasing concentrations of azadirachtin in the diet. Preliminary histological observations indicated that the oviposition decrease was the result of a block in ovarian activity. Additional tests confirmed that azadirachtin induced sterility in the females, even when the flies were exposed for shorter periods (24 h).

Sublethal concentrations of azadirachtin affect food intake, conversion efficiency and feeding behaviour of Spodoptera littoralis (Lepidoptera: Noctuidae)

AbstractSublethal concentrations of azadirachtin incorporated into artificial diet and offered to third-instar larvae of Spodoptera littoralis (Boisduval) prolonged larval instars and reduced food intake. These effects were observed after the treated larvae had been transferred to plain diet; the reduced food intake was therefore a secondary antifeedant effect. Azadirachtin did not influence digestion efficiency but diminished the ability of the larvae to convert both ingested and digested nutrients into growth, particularly immediately after treatment. This effect declined with time once the treatment with azadirachtin had ceased. Growth was more severely reduced than food intake, and the reduction in growth also occurred during periods when food intake was not affected, possibly due to post-ingestive effects.

Differential cytotoxic effects of azadirachtin on Spodoptera frugiperda and mouse cultured cells

AbstractAzadirachtin is a natural biopesticide extracted from the neem tree (Azadirachta indica); however, its safety to mammals is not well documented. This study examined the influence of azadirachtin on the proliferation and morphology of Spodoptera frugiperda insect cells (SF‐9), and mouse erythroleukemia cells (MEL‐GM 86). Cell kinetic studies conducted at 24 h intervals for 144 h showed that azadirachtin treatments (0.05, 0.20 and 0.50 μg/ml) inhibited cell multiplication of the SF‐9 cells. This inhibitory effect was dose dependent. Conversely, azadirachtin at 30 μg/ml did not reduce MEL‐GM 86 cell proliferation. SF‐ 9 cells displayed an increase in cell swelling and cell abnormalities after 48 h of treatment with azadirachtin, whereas no such changes were detected in MEL‐GM 86 cells. A scanning electron microscope study revealed that treatment with an azadirachtin concentration as low as 0.10 μg/ml induced cell swelling and caused distortions on the cell surface ultrastructure of SF‐9 cells characterized by blebbing and holes after 48 h of treatment. However, these effects were not seen in the azadirachtin‐treated MEL‐ GM 86 cells.

Callus cultures ofAzadirachta indica and their potential for the production of Azadirachtin

The content of azadirachtin, an insecticidally active ingredient in neem tree [Azadirachta indica (A. Juss.)] seeds, varies considerably due to environmental factors and for genetic reasons. Furthermore, the tree does not grow in moderate climates. It seems appropriate, therefore, to employ tissue culture techniques for the production of azadirachtin in order to obtain constant amounts of standardized quality. We investigated if and how azadirachtin contents of callus cultures derived from various cell lines are affected by different nutrient media and carbohydrate concentrations. The azadirachtin contents of the calli were analyzed by TLC and HPLC. Azadirachtin contents of callus cultures varied depending on the cell line, the nutrient medium and the carbohydrate source employed.

Effect of neem (Azadirachta indica A. Juss) seed kernel extracts on the larval parasitoid Bracon brevicornis Wesm. (Hym., Braconidae)

AbstractThe alcoholic and hexane extracts of 17 neem ecotypes of India have been found to be toxic to the egg, larval and pupal stages of the cosmopolitan, external gregarious larval parasitoid Bracon brevicornis (Wesm.). In general, the hexane extracts showed higher toxicity against the egg and pupal stages whereas the alcoholic extracts were more toxic against the larvae. Azadirachtin content of the neem ecotypes revealed no apparent correlation with the observed toxicity against different stages of the parasitoid.